Designing of spider silk proteins for human induced pluripotent stem cell-based cardiac tissue engineering.
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ABSTRACT:
SUBMITTER: Esser TU
PROVIDER: S-EPMC8209670 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cardiac tissue engineering is a promising approach to treat cardiovascular diseases, which are a major socio-economic burden worldwide. An optimal material for cardiac tissue engineering, allowing cardiomyocyte attachment and exhibiting proper immunocompatibility, biocompatibility and mechanical characteristics, has not yet emerged. An additional challenge is to develop a fabrication method that enables the generation of proper hierarchical structures and constructs with a high density of cardiomyocytes for optimal contractility. Thus, there is a focus on identifying suitable materials for cardiac tissue engineering. Here, we investigated the interaction of neonatal rat heart cells with engineered spider silk protein (eADF4(C16)) tagged with the tripeptide arginyl-glycyl-aspartic acid cell adhesion motif RGD, which can be used as coating, but can also be 3D printed. Cardiomyocytes, fibroblasts, and endothelial cells attached well to eADF4(C16)-RGD coatings, which did not induce hypertrophy in cardiomyocytes, but allowed response to hypertrophic as well as proliferative stimuli. Furthermore, Kymograph and MUSCLEMOTION analyses showed proper cardiomyocyte beating characteristics on spider silk coatings, and cardiomyocytes formed compact cell aggregates, exhibiting markedly higher speed of contraction than cardiomyocyte mono-layers on fibronectin. The results suggest that eADF4(C16)-RGD is a promising material for cardiac tissue engineering.
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Project description:Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promoted axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.
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Project description:Safety is the foremost issue in all human cell therapies, but human induced pluripotent stem cells (iPSCs) currently lack a useful safety indicator. Studies in chimeric mice have demonstrated that certain lines of iPSCs are tumorigenic; however a similar screen has not been developed for human iPSCs. Here, we show that in vitro cartilage tissue engineering is an excellent tool for screening human iPSC lines for tumorigenic potential. Although all human embryonic stem cells (ESCs) and most iPSC lines tested formed cartilage safely, certain human iPSCs displayed a pro-oncogenic state, as indicated by the presence of secretory tumors during cartilage differentiation in vitro. We observed five abnormal iPSC clones amoungst 21 lines derived from five different reprogramming methods using three cellular origins. We conclude that in vitro cartilage tissue engineering is a useful approach to identify abnormal human iPSC lines.
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Project description:Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.
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Project description:Human induced pluripotent stem (iPS) cells are a promising source of autologous cardiomyocytes to repair and regenerate myocardium for treatment of heart disease. In this study, we have identified a novel strategy to enhance cardiac differentiation of human iPS cells by treating embryoid bodies (EBs) with a histone deacetylase inhibitor, trichostatin A (TSA), together with activin A and bone morphogenetic protein 4 (BMP4). Over a narrow window of concentrations, TSA (1 ng/ml) directed the differentiation of human iPS cells into a cardiomyocyte lineage. TSA also exerted an additive effect with activin A (100 ng/ml) and BMP4 (20 ng/ml). The resulting cardiomyocytes expressed several cardiac-specific transcription factors and contractile proteins at both gene and protein levels. Functionally, the contractile EBs displayed calcium cycling and were responsive to the chronotropic agents isoprenaline (0.1 ?M) and carbachol (1 ?M). Implanting microdissected beating areas of iPS cells into tissue engineering chambers in immunocompromised rats produced engineered constructs that supported their survival, and they maintained spontaneous contraction. Human cardiomyocytes were identified as compact patches of muscle tissue incorporated within a host fibrocellular stroma and were vascularized by host neovessels. In conclusion, human iPS cell-derived cardiomyocytes can be used to engineer functional cardiac muscle tissue for studying the pathophysiology of cardiac disease, for drug discovery test beds, and potentially for generation of cardiac grafts to surgically replace damaged myocardium.
| S-EPMC3754471 | biostudies-literature
Project description:The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.
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Project description:Materials based on biodegradable polyesters, such as poly(butylene terephthalate) (PBT) or poly(butylene terephthalate-co-poly(alkylene glycol) terephthalate) (PBTAT), have potential application as pro-regenerative scaffolds for bone tissue engineering. Herein, the preparation of films composed of PBT or PBTAT and an engineered spider silk protein, (eADF4(C16)), that displays multiple carboxylic acid moieties capable of binding calcium ions and facilitating their biomineralization with calcium carbonate or calcium phosphate is reported. Human mesenchymal stem cells cultured on films mineralized with calcium phosphate show enhanced levels of alkaline phosphatase activity suggesting that such composites have potential use for bone tissue engineering.
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Project description:Background Optogenetics, using light-sensitive proteins, emerged as a unique experimental paradigm to modulate cardiac excitability. We aimed to develop high-resolution optogenetic approaches to modulate electrical activity in 2- and 3-dimensional cardiac tissue models derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Methods and Results To establish light-controllable cardiac tissue models, opsin-carrying HEK293 cells, expressing the light-sensitive cationic-channel CoChR, were mixed with hiPSC-cardiomyocytes to generate 2-dimensional hiPSC-derived cardiac cell-sheets or 3-dimensional engineered heart tissues. Complex illumination patterns were designed with a high-resolution digital micro-mirror device. Optical mapping and force measurements were used to evaluate the tissues' electromechanical properties. The ability to optogenetically pace and shape the tissue's conduction properties was demonstrated by using single or multiple illumination stimulation sites, complex illumination patterns, or diffuse illumination. This allowed to establish in vitro models for optogenetic-based cardiac resynchronization therapy, where the electrical activation could be synchronized (hiPSC-derived cardiac cell-sheets and engineered heart tissue models) and contractile properties improved (engineered heart tissues). Next, reentrant activity (rotors) was induced in the hiPSC-derived cardiac cell-sheets and engineered heart tissue models through optogenetics programmed- or cross-field stimulations. Diffuse illumination protocols were then used to terminate arrhythmias, demonstrating the potential to study optogenetics cardioversion mechanisms and to identify optimal illumination parameters for arrhythmia termination. Conclusions By combining optogenetics and hiPSC technologies, light-controllable human cardiac tissue models could be established, in which tissue excitability can be modulated in a functional, reversible, and localized manner. This approach may bring a unique value for physiological/pathophysiological studies, for disease modeling, and for developing optogenetic-based cardiac pacing, resynchronization, and defibrillation approaches.
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Project description:Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium.
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Project description:Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH result in aggregation of the two proteins. Assembly of eADF3 fibers was induced only in the presence of an elongational flow component. Strikingly, eADF4 formed fibers only in combination with eADF3. On the basis of these results, we propose a model for dragline silk aggregation and early steps of fiber assembly in the microscopic regime.
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